Lipids of Acanthamoeba castellanii. Composition and effects of phagocytosis on incorporation of radioactive precursors.

The lipids of Acanthamoeba castellanii (Neff) consist of 52% neutral lipids and 48% polar lipids. Triglycerides account for 75% and free sterols for 17% of the neutral lipids. The major phospholipids are phosphatidylcholine (45%), phosphatidylethanolamine (33%), phosphatidylserine (10%), a phosphoinositide (6%), and diphosphatidylglycerol (4%). The phosphoinositide is unique in that it contains fatty acids, aldehyde, inositol, and phosphate in the ratio of 1.4:0.5:1.1, but it contains no glycerol. Sphingomyelin, cerebrosides, psychosine, and glycoglycerides were not detected, but small amounts of unidentified long chain bases and sugars are present. The rates of uptake of palmitate-1-(14)C and of its incorporation into glycerides and phospholipids were not affected by the phagocytosis of polystyrene latex beads. Although phagocytosis usually decreased the uptake by amebas of phosphate-(32)P, serine-U-(14)C, and inositol-2-(3)H, their subsequent incroporation into phospholipids was not demonstrably stimulated or inhibited by phagocytosis. Phagocytosis did seem to increase the incorporation into ameba phospholipids of phosphatidylcholine-1 ,2-(14)C but not that of phosphatidylethanolamine-1 ,2-(14)C. These experiments, in which the incorporation of radioactive precursors into total cell lipids was measured, do not, of course, eliminate the possibility that localized effects may occur.

Plasma and phagosome membranes of Acanthamoeba castellanii.

Plasma membranes were isolated from the ameba Acanthamoeba castellanii by low-speed velocity centrifugation followed by equilibrium centrifugation in a sucrose gradient. The isolated membranes had a high ratio of sterol to phospholipid (0.98 moles/mole) and of phospholipid to protein (0.43 mg/mg). The plasma membranes had very low concentrations of DNA, RNA, lipid inositol, and glycerides. Glycolipids and glycoproteins were enriched in the plasma membranes relative to their concentrations in the whole cell. The plasma membranes were also judged to be of high purity by the absence, or very low level, of enzymatic activities considered to be indicative of other cell membranes, and by electron microscope examination. Alkaline phosphatase and 5'-nucleotidase activities were enriched in the plasma membranes 13-fold relative to the whole homogenate and had higher specific activities in the plasma membranes than in any other cell fractions. A Mg(++) adenosine triphosphatase (ATPase) was enriched sixfold in the plasma membranes relative to the whole homogenate. The phospholipids of the plasma membranes contained more phosphatidylethanolamine and phosphatidylserine and less phosphatidylcholine than did the phospholipids of the whole cells. There were differences in the fatty acid compositions of corresponding phospholipids in the plasma membranes and whole cells but no difference in the ratios of total saturated to unsaturated fatty acids. The membranes of phagosomes isolated from amebae that had ingested polystyrene latex had essentially the same phospholipid, sterol, and enzymatic composition as plasma membranes.

Biochemical studies on pulmonary response to inhalation of methylene chloride.

Biochemical response to the toxic lung damage induced by inhalation of methylene chloride was studied. Significant increases in protein, hexose, sialic acid, lactate dehydrogenase, acid and alkaline phosphatase content were observed in the cell-free lavage effluents from lungs of exposed rats compared to the control animals. This was interpreted as increased cell damage accompanied by enhanced pulmonary secretions, perhaps of glycoproteins and mucins, as a result of inhalation toxicity.

Hyaluronic acid--an indicator of pulmonary injury?

Hyaluronic acid was the only glysosaminoglycan detected in the pulmonary secretions of healthy adult rats exposed to inhalation to methylene chloride, but not of control animals. The compound migrated as a single spot with the mobility of standard hyaluronic acid on cellulose acetate electrophoresis and disappeared after digestion with testicular hyaluronidase. Its identification was confirmed by finding hexuronate/hexosamine in a molar ratio of approx. 1. Glucosamine represented over 97% of the total hexosamine, the remaining 3% being galactosamine. No hexose or sulfate could be detected. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis showed no protein associated with this glycosaminoglycan. It appears that the secretion of hyaluronic acid into the airways may be the result of pulmonary inflammation induced by the toxic effects of methylene chloride.

Experimental hexachlorophene intoxication in young swine.

Hexachlorophene (HCP) intoxication in swine was studied as a possible model of HCP poisoning in human beings. Swine were topically or orally given HCP each day, and observations were made on central nervous system function, hematologic measurements, serum chemical analyses, necropsy, microscopic examination of tissues, and concentrations of HCP residues in blood and tissue. Intoxication was observed in orally exposed swine, but the syndrome was not consistently produced in topically exposed swine. Signs and microscopic lesions were all related to dysfunction of the central nervous system, e.g., incoordination, paresis, and status spongiosus. Occurrence of signs and lesions, mortality rates, and concentrations of HCP residue were positively correlated with the dosage of HCP given.

Identification of pY654-ß-catenin as a critical co-factor in hypoxia-inducible factor-1α signaling and tumor responses to hypoxia.

Hypoxia is linked to epithelial-mesenchymal transition (EMT) and tumor progression in numerous carcinomas. Responses to hypoxia are thought to operate via hypoxia-inducible factors (HIFs), but the importance of co-factors that regulate HIF signaling within tumors is not well understood. Here, we elucidate a signaling pathway that physically and functionally couples tyrosine phosphorylation of ß-catenin to HIF1α signaling and HIF1α-mediated tumor EMT. Primary human lung adenocarcinomas accumulate pY654-ß-catenin and HIF1α. All pY654-ß-catenin, and only the tyrosine phosphorylated form, was found complexed with HIF1α and active Src, both within the human tumors and in lung tumor cell lines exposed to hypoxia. Phosphorylation of Y654, generated by hypoxia mediated, reactive oxygen species (ROS)-dependent Src kinase activation, was required for ß-catenin to interact with HIF1α and Src, to promote HIF1α transcriptional activity, and for hypoxia-induced EMT. Mice bearing hypoxic pancreatic islet adenomas, generated by treatment with anti-vascular endothelial growth factor antibodies, accumulate HIF1α/pY654-ß-catenin complexes and develop an invasive phenotype. Concurrent administration of the ROS inhibitor N-acetylcysteine abrogated ß-catenin/HIF pathway activity and restored adenoma architecture. Collectively, the findings implicate accumulation of pY654-ß-catenin specifically complexed to HIF1α and Src kinase as critically involved in HIF1α signaling and tumor invasion. The findings also suggest that targeting ROS-dependent aspects of the pY654-ß-catenin/ HIF1α pathway may attenuate untoward biological effects of anti-angiogenic agents and tumor hypoxia.

Formaldehyde and hepatotoxicity: a review.

Exposure to formaldehyde appears to be associated with hepatoxicity in many species, including humans, following injection, ingestion, or inhalation. Macroscopic, microscopic, and biochemical manifestations in the liver include alterations in weight, centrilobular vacuolization, focal cellular necrosis, and increased alkaline phosphatase concentrations. Time-related changes in the pattern of the effects are suggested as one goes from acute exposure by inhalation at greater concentrations to repeated exposure at lesser concentrations. Although the hepatic changes are generally not extensive and can be reversible following acute exposure, the potential exists for them to progressively become more serious with repeated exposures. There are several possible mechanisms for the toxicity. Depending on the route of exposure could include direct effects on hepatocytes and/or indirect effects through the circulatory and immune systems. The catabolism of formaldehyde includes conversion to CO2 by reactions involving glutathione. Many hepatotoxic chemicals require glutathione for detoxification. Formaldehyde may then have the potential to cause additive toxicity with such chemicals in some circumstances.

Hexachlorophene lesions in newborn infants.

Vacuolization of the white matter of the brain is produced by a number of disease entities and chemicals, including hexachlorophene. Brains of 135 stillborn infants and infants dying in the neonatal period were examined for vacuole formation in the white matter to determine if any hexachlorophene-like lesions could be found. A nonsignificant excess of vaculoes was found in infants bathed in hexachlorophene at birth compared with infants not bathed in it. Analysis of 11 brains for hexachlorophene showed that detectable levels were present in five, all of which showed vacuolization. None was detected in the remaining six, three of which also showed vacuolization. In two of these there was no hexachlorophene exposure.

Appraisal of existing methodology in aspiration toxicity testing.

New methods or modifications of currently used methods in aspiration toxicology must be capable of accurately assessing the hazard and toxicity potentials of those products which are capable of being aspirated. The methods evaluated in the rat were intratracheal injections,intravenous injections, and modifications of the Gerarde technique. A product containing petroleum distillates and a standard viscosity petroleum distillate sample were administered to Osborne-Mendel rats, rabbits, and guinea pigs in ml/kg doses. The utility of the methods was determined by comparing lung weight increases, lung-body weight ratios, gross pathological lung changes, and mortality. The modified Gerarde technique showed the best potential for predicting aspiration hazard and toxicity based on the severity of the reactions which, using a radiolabeled oil, was shown to be a function of the amount of product which enters and reacts with the lungs. The influence of preventing the swallowing reflex to assure aspiration of the oil was assessed in the rat. The results show that less than 10% of the dose is aspirated when the swallowing reflex occurs. Methods of sacrifice were investigated for an effect on rat lung weight. No tested method except stunning with cervical dislocation produced an effect. Tests were conducted on rats and rabbits to determine differences in lung reactivity to the influence of inhalation anesthetics, pentothal sodium, or no anesthesia in response to petroleum distillate administration and to determine which test species is the better animal model. The resuls indicate that the rat is the better test model because of degree of lung reaction, size, and economy, and that inhalation anesthesia with ether is preferable over the other choices.

Percutaneous absorption of radiolabeled TRIS from flame-retarded fabric.

Surface tris-(2,3-dibromopropyl) phosphate (TRIS) was removed from flame-retarded polyester fabric by benzene-hexane extraction and replaced with 14C-TRIS. Sections of the radiolabeled fabric (10 X 12 cm) were placed in contact with the clipped skins of rabbits, and urine and feces were collected over a 96 hr period. The cloths were allowed to remain dry or were moistened with either urine or simulated sweat. A similar collection of urine and feces was performed following i.v. injection of 14C-TRIS. From the results of these experiments, it can be calculated that up to 17 percent of the radiolabel on the cloth penetrated rabbit skin over the 96 hr period of exposure. Most of the excreted radiolabel appeared in the urine, with the kidney being the organ of highest specific radioactivity. The highest absorption of radiolabel occurred in the urine-moistened cloth group. Moistening the cloth with simulated sweat produced no increase in absorption over that seen with dry cloth.

Bioavailability in rabbits of formaldehyde from durable-press textiles.

Carbon-14-labeled formaldehyde was used per se, or was used in the synthesis of dimethyloldihydroxyethyleneurea (DMDHEU), which was incorporated into cotton or cotton/polyester blend fabric. Patches of the fabric containing known quantities of radioactive DMDHEU were applied to the backs of New Zealand White rabbits for periods up to 48 h. The rabbits were placed in specially constructed metabolism chambers designed to prevent either inhalation of volatile material emanating from the fabric or interference of any volatile material from the fabric with trapping of expired carbon dioxide. The results of the studies indicate that aqueous formaldehyde covered with a latex barrier is absorbed and retained in the layers of skin in direct contact with the formaldehyde. Approximately 65% of a dose of [14C] formaldehyde was recovered in skin 4 h after application. Skin samples from the backs of rabbits to which durable-press fabric prepared from radiolabeled DMDHEU had been applied were found to have 0.09-2.61% of the total 14C contained in the cloth patches. The levels of radioactivity recovered from the skin varied with degree of occlusion of the cloth, presence or absence of perspiration, type of synthesis used for the preparation of DMDHEU, and whether cotton or cotton/polyester blend fabric was used. Other tissues and organs had only low levels of radioactivity. Injected [14C] formaldehyde was rapidly expired as 14CO2 (28.6% of the dose within 4 h; 37.0% within 48 h). Metabolism and distribution of formaldehyde was found to be dependent on route of administration: i.e., topical application resulted in high skin levels, whereas intravenous injection led to rapid pulmonary and renal excretion and retention of radioactivity in liver, kidney, and blood.